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EP0366451A2 - Utilisation des polyamides oxydés, en particulier NN'-Bis-(3-propionaldéhyde)-1-4-diaminobutane (spermine dialdéhyde) comme agents immunosuppresseurs - Google Patents

Utilisation des polyamides oxydés, en particulier NN'-Bis-(3-propionaldéhyde)-1-4-diaminobutane (spermine dialdéhyde) comme agents immunosuppresseurs Download PDF

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EP0366451A2
EP0366451A2 EP89311013A EP89311013A EP0366451A2 EP 0366451 A2 EP0366451 A2 EP 0366451A2 EP 89311013 A EP89311013 A EP 89311013A EP 89311013 A EP89311013 A EP 89311013A EP 0366451 A2 EP0366451 A2 EP 0366451A2
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cells
alk1
independently
alkylene
alk2
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EP0366451B1 (fr
EP0366451A3 (fr
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Catherine Y. Lau
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Ortho Pharmaceutical Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • C07C217/40—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines having at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the same carbon atom of the carbon skeleton, e.g. amino-ketals, ortho esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C223/00—Compounds containing amino and —CHO groups bound to the same carbon skeleton
    • C07C223/02—Compounds containing amino and —CHO groups bound to the same carbon skeleton having amino groups bound to acyclic carbon atoms of the carbon skeleton

Definitions

  • This invention relates to the use of oxidized polyamines, and especially aminoaldehydes such as NN′-Bis-(3-propionaldehyde)-1-4-diaminobutane (spermine bisaldehyde) both in vitro and in vivo to elicit an immunosuppressive response in living cells. It also relates to the therapeutic application of these compounds to induce an immunosuppressive response in a living organism.
  • aminoaldehydes such as NN′-Bis-(3-propionaldehyde)-1-4-diaminobutane (spermine bisaldehyde)
  • Polyamines such as spermine, spermidine and putrescine are widely distributed in mammalian cells, although they are found to differ in their relative concentrations. Oxidized Polyamines are believed to inhibit growth of parasites (D.M.L. MORGAN and J.R. CHRISTENSEN, Adv. Polyamine Res., 4, 169-174 (1983); D.M.L. MORGAN, U. BACHRACH, Y.G. ASSARAF, E. HARARI and J. GOLENSER, Biochem. J., 236, 97-101 (1986)), suppress infectivity of selected strains of phage and bacteria (U. BACHRACH, S. DON and H. WIENER, J. Gen.
  • Oxidized Polyamines are believed to inhibit growth of parasites (D.M.L. MORGAN and J.R. CHRISTENSEN, Adv. Polyamine Res., 4, 169-174 (1983); D.M.L. MORGAN, U. BACHRACH
  • Oxidation of spermine by PAO revealed six major oxidation products in addition to ammonia and hydrogen peroxide (R.S. LABIB and T.B. TOMASI, JR., Eur. J. Immunol., 11, 266-269 (1981)).
  • the inhibitory effect of each of the Products has not been analyzed but it was believed that the dioxidized spermine or spermine dialdehyde (NN′-Bis-(3-propionaldehyde) -1-4-diaminobutane) would demonstrate activity.
  • Mouse spleen and bone marrow cells were incubated separately with different concentrations of spermine dialdehyde in vitro for 1 hour. Cells were washed extensively and set up in a Con A stimulated proliferation assay. A graph is depicted wherein percentage response is plotted as a function of various concentrations of spermine dialdehyde.
  • C57BL spleen and bone marrow cell mixtures were treated with different concentrations of spermine dialdehyde or control preparation and injected i.v. into lethally irradiated AKR mice.
  • a graph is depicted wherein percentage survival is scored as a function of time.
  • mice C57BL mice were injected i.p. daily with different doses of spermine dialdehyde. On day 2, they also received 107 DBA spleen cells. Seven days later, animals were sacrificed and percentage of cytotoxic T cells (measured by % specific lipes generated in the spleen was calculated and plotted as a function of concentration of spermine dialdehyde.
  • BAH/C (H2 d ) skin grafts were transplanted into C3H mice.
  • Spermine dialdehyde was administered S.C. right after transplantation, and continued throughout the study.
  • Cyclosporin A was run as a positive control.
  • % skin graft survival was plotted versus days post transplantation.
  • Delayed type hypersensitivity was induced in the footpad of B6D2F1 mice and swelling measured in mm.
  • Spermine dialdehyde was administered i.p. and i.m. at various timeperiods.
  • Splenic cells were treated with various concentrations of SDA or an analogue for 30 min. at 37°C. Cells were washed and cultured in the presence of Concanavalin A for 3 days. [3H] Thymidine 0.1 ⁇ Ci/well was added and cells were harvested after 18-24 hrs. SDA was the most potent antiproliferation drug, with an IC50 of 0.70 ⁇ M. Although butyl dialdehyde was 30 fold less potent than SDA, it should be metabolically more stable and hence clinically more relevant.
  • Murine leukemic L1210 cell supernatants were incubated with different concentrations of SDA or butyl dialdehyde, and AdoMet DC activity was estimated by monitoring the release of [14C] CO2. Inhibition curves for both drugs were superimposable with IC50 values of 0.12-0.13 x 10 ⁇ 3 M.
  • the figure illustrates the comparative profile of the inhibition of key regulatory enzymes of polyamine synthesis by SDA, its analogue, butyl dialdehyde (BDA), difluoromethyl orinithine (DFMO) and methyl glyoxal bis (guanylhydrazone) (MGBG).
  • DFMO inhibited ODC with high potency, while MGBG selectively reduced AdoMet IC activity.
  • the analogue BDA was similar to MGBG in its selectivity to AdoMet DC.
  • SDA was the only drug that inhibited both of the key enzymes, thereby eliminating the compensatory response (of DFMO on AdoMet DC) and ensuring complete inhibition of polyamine biosynthesis.
  • the present invention provides a method for inducing an immunosuppressive response in living cells which comprises the administration to those cells of an effective amount of a substantially pure form of a compound having the General Formula: OCH - ALK1 - NR2 - CH2 - AlK2 - CH2 -NR2 - Z wherein ALK1 is independently alkylene; R2 is independently hydrogen or -CH2R3; R3 is independently alkyl; ALK2 is alkylene; Z is H or ALK1-CHO; or the acid addition salt thereof.
  • ALK1 is independently alkylene
  • R2 is independently hydrogen or -CH2R3
  • R3 is independently alkyl
  • ALK2 is alkylene
  • Z is H or ALK1-CHO; or the acid addition salt thereof.
  • This immunosuppressive response is particularly selective for the suppression of the proliferation of T cell populations, especially the helper T cell and cytotoxic T cell subpopulations.
  • Also provided are methods for inducing an immunosuppressive response in a living organism which comprises the administration of a compound having the above formula to said organism, in substantially pure form and in an amount effective to induce said response.
  • the methods as described herein are particularly suitable in the therapeutic control of various immunologically related disease states such as graft vs. host rejections, delayed hypersensitivity and the like.
  • the present inventors have discovered that compounds, particularly those obtained by synthetic methods, having the General Formula: OCH - ALK1 - NR2 - CH2 - AlK2 - CH2 - NR2 - Z wherein ALK1 is independently alkylene; R2 is independently hydrogen or -CH2R3; R3 is independently alkyl; ALK2 is alkylene; Z is H or ALK1-CHO; or the acid addition salt thereof.
  • [ General Formula I ] are useful in inducing an immunosuppressive response in living cells when applied to those cells in substantially pure form.
  • ALK1 ALK2, and R3 are each independently straight or branched chain alkylene of about 1 to about 8 carbons, preferably about 1 to about 6 carbons, more preferably about 1 to 4 carbon atoms, and most preferably about 1 to about 2 carbon atoms.
  • R2 is hydrogen.
  • Z is ALK1-CHO1
  • R2 is hydrogen
  • ALK1 and ALK2 are each independently about 1 to about 6 carbon atoms, preferably about 1 to about 4 carbon atoms, most preferably about 1 to about 2 carbon atoms.
  • spermine dialdehyde may be mentioned.
  • the presently described compounds are also useful in the therapeutic inducement of an immunosuppressive response in a living organism.
  • one of the compounds, synthetic spermine dialdehyde, used according to the present invention has demonstrated enhanced efficacy and a substantially reduced toxicity [not lethal up to 400 mg/kg, contrary to what was reported in the literature (LD100 40 mg/kg, Israel (1973) Supra)].
  • the use of these molecules, particularly spermine dialdehyde, both in in vitro applications as well as in vivo applications to induce an immunosuppressive response is therefore described in detail herein.
  • the term "immunosuppressive response" refers to a suppression in the proliferation of cell types originating from hemopoietic stem cells, particularly the lymphoid lineage.
  • the use of the compounds as described herein is particularly suitable in the selective suppression of the T-cell population, particularly the T-cytotoxic, T-helper subpopulations.
  • the immunosuppression may also be described as antigen nonspecific, which is exemplified by a 99% suppression of mitogen-induced T cell proliferation. In some cases, contrary to the art, this selective suppression has been shown to be irreversible.
  • the compounds for use as described herein may be obtained in any convenient manner.
  • the compounds are preferably used in "substantially pure form" which means substantially free of enzymes or other agents that might be present during its formation, and that will interfere with its efficacy or that might increase toxicity.
  • the compounds are preferably 95% pure, more preferably 99% pure, and most preferably 100%, as measured by nuclear magnetic resonance, mass spectro analysis, and HPLC. Inert materials may be present in trace amounts.
  • the compounds may be obtained by enzymatic oxidation of naturally occurring polyamines by techniques known to the art, as long as the product is purified from the enzymes using conventional purification means such as chromatography and the like.
  • Suitable enzymes for use in an enzymatic oxidation process are those effective to bring about the rapid oxidative deamination of the natural polyamine.
  • Illustrative of such enzymes are amine oxidases obtained from ruminant sera such as beef serum, sheep serum, fetal calf serum and the like. Also applicable are oxidases obtained from mouse amniotic fluid, human pregnant sera, and the like.
  • compounds for use herein are amino dialdehydes.
  • the dialdehyde compound is synthesized by conversion of a diacetal in the presence of an acid.
  • Preferred diacetals are represented by Formula: wherein R1 is independently alkyl or benzyl ALk1 is independently alkylene; R2 is independently hydrogen or -CH2R3 R3 is independently alkyl; and ALk2 is alkylene,
  • a chloroaldehyde of Formula (II) in acetal form is reacted with potassium phthalimide of Formula (III) in equal molar amounts at an elevated temperature to yield the phthalimide of Formula (IV).
  • Examples of the compound of Formula (II) are 3-chloroacetaldehyde diethyl acetal and 3-chloro propionaldehyde diethylacetal.
  • the amine is then released by reaction with hydrazine at an elevated temperature, yielding the primary amine of Formula (V).
  • the primary amine (V) can then be taken on as shown in Reaction Scheme II or can be modified to produce a secondary amine.
  • the primary amine of Formula (V) is reacted with an anhydride such as formic-acetic anhydride or acetic anhydride at temperatures that can vary from about 0°C to about room temperatures to yield the amide of Formula (VI).
  • the amide is then reduced with lithium aluminum hydride in tetrahydrofuran at reflux to yield the secondary amine of Formula (VII).
  • R1, ALK1 and R3 are as defined for Formula (X) above.
  • the corresponding diethyl compound may be dissolved in a solvent and reacted with benzyl alcohol in the presence of a small amount of acid such as trichloracetic acid followed by distillation to drive off the ethanol byproduct.
  • the diethyl compound may be dissolved in the alcohol, e.g. methanol for the dimethyl acetal, and reacted as in the benzyl case.
  • the primary or secondary amine designated as compound (V) or (VII), respectively, is reacted with a diacid chloride of Formula (VIII).
  • diacid chlorides are succinyl chloride and glutaryl chloride.
  • the reaction is conducted at a low temperature of about -70° to -20° in the presence of a mild base such as triethylamine to yield the diamide of Formula (IX).
  • the diamide (IX) is then reduced with a reducing agent such as lithium aluminum hydride in tetrahydrofuran at reflux to yield the diamine diacetal of Formula (X).
  • the diamine diacetal (X) can be stored for extended periods and brought to the site of administration to a patient for the conditions described above.
  • the final product is preferably purified by making the crystalline salt form of the diamine (X) with an acid.
  • the free amine can then be resynthesized from the salt.
  • storage may be easier with the salt form. It should be noted that creation of the salt with acid must be carried out carefully since excessive amounts of the acid will destroy the acetal moieties.
  • the diacetal (X) would be reacted with an acid such as hydrochloric acid to yield the free dialdehyde.
  • an acid such as hydrochloric acid
  • Sodium hydroxide may be used to regenerate (X) from the salt form, if desired.
  • the thus-produced mixed carboxylic acid-amide is then reacted with a mole of a different amine of formula (V) or (VII) using a peptide coupling reagent such as (2-ethoxy-1-ethoxycarbonyl-­1,2-diahydroquinoline, known as EEDQ, or 1,3-dicyclohexylcarbodiimide, known as DCC, to yield an amide of formula (IX) wherein the two ALK1 moieties are not the same.
  • a peptide coupling reagent such as (2-ethoxy-1-ethoxycarbonyl-­1,2-diahydroquinoline, known as EEDQ, or 1,3-dicyclohexylcarbodiimide, known as DCC
  • concentrations of oxidized polyamines suitable for use herein are concentrations effective to induce the desired immunosuppressive response without exhibiting substantial cell toxicity. Effective concentrations may vary widely according to the particular cells it is desired to imnunosuppress, and the organism from which these cells are obtained. Exemplary concentrations for use herein may be extrapolated from the preferred concentrations for spermine dialdehyde These concentrations generally range from about 0.01 mM to about 0.2 mM, more preferably about 0.03 mM to about 0.1 mM, and most preferably about 0.03 mM to about 0.06 mM.
  • the immunosuppressive response induced by the techniques described herein, and the efficacy of the dosing, may also be measured in vitro by methods conventional to the art. This may be accomplished by the incubation of cells to be immunosuppressed with various concentrations of the compound, and comparison of the ensuing proliferation of these cell populations with that of control samples that have not been treated with the compound as described herein.
  • the oxidized polyamines are administered to the cells in any suitable physiologically compatible vehicle such as saline, phosphate buffer saline, methycellulose solutions, and the like. Solutions of the compound or homogeneous dispersions are preferred for such administration.
  • the cells are allowed to incubate with the compound in a suitable growth medium such as minimum essential medium (MEM), RPMI 1640 and other suitable tissue culture media, to maintain cellular viability.
  • MEM minimum essential medium
  • RPMI 1640 RPMI 1640 and other suitable tissue culture media
  • This incubation generally takes place for a period of time sufficient to induce suppression of cell proliferation of at least about 25%, and preferably at least about 50%, as compared to control values of cellular growth.
  • the incubation generally ranges for a period of time of about 10 minutes to about 1 hour, preferably about 10 minutes to about 30 minutes.
  • spermine dialdehyde Treatment with the spermine dialdehyde can inactivate T lymphocytes without exhibiting toxic effects on the marrow cells themselves.
  • This phenomenum has been shown to alleviate typical graft vs. host reactions, which are noted as being lethal to patients undergoing bone marrow transplantations.
  • Suitable treatment parameters for such bone marrow extracts in human subjects may be the ex vivo treatment of cells as described above.
  • the present inventors have also discovered that direct in vivo administration of the compounds may also be effective to elicit an immunosuppressive response in an organism.
  • in vivo administration of the compound spermine dialdehyde can greatly reduce graft vs. host reactions in a living organism.
  • administration of this compound will suppress the generation of cytotoxic T cells. It is believed that cytotoxic T cells play a role in the mediation of organ graft rejections, and thus, in vivo suppression of these cells will lead to prolonged graft survival.
  • the in vivo administration of these compounds has far-reaching therapeutic advantages.
  • RA Rhematoid Arthritis
  • Compounds administered therapeutically according to the method of the invention may be prepared as described above, and are also preferably dialdehydes synthesized from a diacetal.
  • Formulations containing the oxidized polyamines or pharmaceutically acceptable salts thereof may also be used in this method, including ingredients such as conventional pharmaceutically acceptable carriers, like saline or sterile water, or ingredients to aid solubility or preservation of the formulation.
  • the preferred in vivo mode of administration of the compounds according to the method of this invention to achieve the desired immunosuppression is parenteral, more preferably intravenous or subcutaneous, and most preferably subcutaneous. It is not believed, however, that the specific mode of administration is critical to the practice of the present method so long as an effective amount of the compound enters the blood stream.
  • the present inventors have found that about 5-10 mg/kg of spermine dialdehyde administered intraveneously or subcutaneously was effective to suppress graft vs. host reactions, as detailed in the Examples section.
  • Subcutaneous dosing of about 10 mg/kg i.p. - 20 mg/kg i.p. correlated well with the i.v. dosing.
  • subcutaneous administration those skilled in the art would recognize the necessity to increase the dose. Accordingly, the only practical limits are dictated by optimum efficacy, and hence, all such doses, frequencies of administration, and modes of administration are intended to be included within the subject method.
  • Efficacy of the method of the invention may be determined by observations of the clinical manifestations of graft vs. host reactions (either in the human or in one or more animal species) such as hunched back, diarrhea, alopecia, poor physical condition, wasting, and in the most drastic cases, death. Reduction of these symptoms would be an indication that the compound is exerting its effect, at the chosen dosing. Signs of toxicity for example, leukopenia, anemia, lack of reconstitution, and the like would indicate that the dosage should be reduced.
  • Indirect measurement of efficacy may also be useful in establishing and monitoring dosage levels. For example, this may be determined by assaying the cytotoxic T cell populations by mixed lymphocyte reaction assays, using peripheral T cells.
  • ALDH The involvement of ALDH was obvious with the following observations: 1) NAD linked cytosolic ALDH could oxidize SDA to a presumably nontoxic product (spermic acid); 2) murine leukemic cells L1210/CPA and L1210/0, having different levels of ALDH differ in their sensitivity to SDA and 3) the ALDH inhibitor, DEAB, potentiated the antitumor and lethal effect of SDA on L1210/CPA cells and on specific colonies of hemopoietic progenitor cells.
  • SDA could be used as a high affinity substrate for cytosolic ALDH in both liver and tumor cells.
  • the known inhibitors, disulfiram, DEAB, and acrolein inhibited ALDH with SDA as substrate, consistent with previous reports on ALDH using other aldehydes.
  • the potent inhibition by acrolein may be particularly important in toxicities associated with SDA treatment.
  • SDA Based on the chemical dynamics of the SDA molecule in solution and its comparison with the metabolism of cyclophosphamide, it can be postulated that SDA could give rise to acrolein by the process of ⁇ -elimination under favourable, base catalytic conditions. If acrolein produced by metabolism of SDA were to inhibit ALDH, there would be an increase in the formation of acrolein and an increase in the chances of unrelated toxic effects.
  • ALDH activity has been shown to be the mechanism of antitumor drug resistance to cyclophosphamide in two experimental murine leukemia cell lines (see above-cited references).
  • ALDH activity was 200 fold higher in L1210/CPA cells compared to wild type sensitive L1210/0 cells.
  • proliferation assays showed that L1210/0 cells were only 2 fold more sensitive than L1210/CPA cells.
  • DEAB pretreatment resulted in equisensitivity for both cell types.
  • Murine bone marrow cell proliferation based on [3H]-thymidine incorporation, indicated relatively less cytotoxicity at lower concentrations of SDA; DEAB pretreatment, however, caused dramatic enhancement of SDA action. It may be pointed out that the influence of ALDH on SDA activity would be relevant only at lower concentrations ( ⁇ 20 ⁇ M). At higher concentrations of SDA, the nonspecific toxicity of the drug appears to predominate, irrespective of intracellular ALDH levels. A good correlation was observed between proliferation and appearance of hemopoietic progenitor colonies with respect to SDA dosage. In agreement with previous reports on cyclophosphamide, BFU-E and CFU-GM were found to be more sensitive to SDA in the presence of an ALDH inhibitor.
  • Bone marrow toxicity and tumor cell or immune responsive T lymphocyte resistance are the two major limiting factors in bone marrow transplantation.
  • Identification of different ALDH isozymes (in hemopoietic cells and contaminating cells) using specific immunological probes and inhibitors would facilitate the development of optimal conditions to selectively sensitize the uneconomic species to SDA and thereby possibly offer an enhanced therapeutic margin of safety.
  • Spermine bis-acetal is dissolved in 1N HCl at a concentration of 100 mM (47.5 mg/ml). The solution is incubated in a 37°C water bath for an hour. At the end of the incubation period, the 100 mM solution is diluted to 20 mM with purified water. The pH of the 20 mM solution is adjusted to between 5.0 and 6.5 using 10N NaOH. This pH adjusted 20 mM solution is the stock for all test material.
  • Control vehicle stock solution is obtained by titrating a 0.2M HCl solution with 10N NaOH and adjusting the pH to between 5.0 and 6.5. To obtain a control working solution, the stock solution is then diluted accordingly as with the test solution.
  • PHA-P Purified phytohemagglutinin
  • PWM Pokeweed mitogen
  • Tritated thymidine and chromium (51Cr) were obtained from New England Nuclear.
  • Con A was obtained from Sigma.
  • C57/B6, DBA/2, C3H and Balb/C mice were obtained from the Jackson Laboratory.
  • ⁇ -MEM ⁇ -minimum essential medium
  • ⁇ -MEM ⁇ -minimum essential medium
  • penicillin Gabco 50 Mg/ml
  • streptomycin Gibco, 100 Mg/ml
  • L-glutamine Gibco, 2.0 mM
  • FCS fetal calf serum
  • Bone marrow cells were obtained from suitable long bones by flushing with a 0.25 gauge needle. Cells were then washed three times with RPMI before use.
  • Spleen cells were prepared by passing the spleen through a fine wire mesh. Cells were resuspended in RPMI and washed three times before use.
  • Peripheral blood lymphocytes were obtained from the blood of normal donors by using standard Ficoll-Hypaque gradient techniques.
  • the T and non-T cells were separated by standard sheep red blood cell (SRBC) rosetting technique. Briefly, 5 x 106/ml PBL were incubated with 1% neuraminidase-treated SRBC, and rosetted cells were separated from non-rosetted cells in a Ficoll-Hypaque gradient. The rosetted cells were designated as T cells.
  • Purified B lymphocytes were obtained from non-rosetted cells by removal of residual T cells using the Pan T monoclonal antibody OKT 11 and complement.
  • T cell proliferation 7 x 104 - 105 human T lymphocytes were cultured in flat bottom microtiter plates (Flow) in the presence of 0.1% PHA-P or 1/64 PWM for 4 days. 3H-thymidine (0.1 MCi/well) was added for the last 6 hours of the culture period.
  • T cell dependent B cell proliferation 4 x 104 T cells and 105 B cells were cultured together in the presence of 1/64 dilution of PWM for 4 days. 3H-thymidine was added for the least 6 hours of the culture period. B cell proliferation was carried out in exactly the same manner with no T cells added to the culture.
  • Mouse spleen cells were cultured for 3 days in flat bottom microtiter relates at 105 cells per well in the presence of 2 mg/ml of Con A. 3H-thymidine (0.1 microcurie/well) was added for the last 4-6 hours of the culture period. Cells were harvested as described above.
  • bone marrow cells from normal donor mice were transplanted to totally histoincompatible and lethally irradiated recipients. All recipient mice received 9.5 Gy (950 rads) total body irradiation on day 0 and were then housed in microisolator cages thereafter. They were given autoclaved rodent laboratory chow throughout the whole study and also were allowed to feed ad libitum. Recipients also received 1.5 mg/kg of gentamicin subcutaneously for 20 days after irradiation. On the day of irradiation, bone marrow cells mixed in 3:1 ratio with spleen cells were removed from donors and incubated for an hour with different concentrations of spermine dialdehyde for 1 hour.
  • GVHD graft vs. host disease
  • graft vs. host reaction in rats was measured by popliteal lymph node enlargement induced by reinjection of parental spleen cells into F1 recipients.
  • F1 rats were divided into groups of 10 and given daily IV injections of different doses of the test compound (spermine dialdehyde) or control preparation. Two days after the first injection, they received 9 x 106 spleen cells from a parental Lewis strain (0.3 ml of a 27 x 106 cell/ml preparation was injected under the skin of the ventral surface of each of the recipient's right hind foot). Injection of test compound and control article continued for 6 more days. On the seventh day, rats were sacrificed by CO2 treatment. Both left and right politeal lymph nodes were removed and cleaned of any adhering tissue. The extent of enlargement was measured by weighing both left and right lymph nodes and differences between the two were calculated.
  • cytotoxic T cells generated in the spleen were quantified by measuring the ability of those cells to lyse 51Cr labelled PB15 target cells (a mastocytoma, cells of DBA origin or Con A stimulated DBA spleen blasts).
  • P815 cells or blasts were suspended at 107 cells/ml and incubated with 200 MCi of 51Cr for 1 hour at 37°C. Cells were then washed 3 times and then mixed with C57BL spleen cells at ratios of 20:1, 10:1 and 5:1 (C57BL:P815) in a V-bottom microlitre plate with a final volume of 200 ml. Cells were spun for 6 minutes and incubated for 4 hours at 37°C. After incubation, 100 Ml of supernatant was removed and counted in the gamma counter. The percentage of cytotoxic T cells was calculated:
  • mice Two pairs of Swiss Webster mice were injected intraperitoneally at 200 mg and 400 mg/kg respectively and observed for toxic effects. Control groups were injected with control preparation. Two weeks after dosing, animals were necropsied and organs were examined for abnormalities.
  • Aldehyde test was performed according to the method described by Sawicki et al., Analytical Chem., 33, 93-96 (1961). Briefly, 50 Ml of 0.4% solution of 3-methyl-2-benzothiazolone hydrazone (MBTH) was added to 50 Ml of the test solution. The mixture was allowed to stand at room temperature for 30 minutes, then 200 Ml of a 0.2% solution of ferric chloride was added and the mixture was then left at room temperature for 10 minutes. Six hundred and fifty Ml of acetone was then added to the sample with slow agitation and the color intensity of the sample was quantitated by spectrophotometric reading at wavelength 670.
  • MBTH 3-methyl-2-benzothiazolone hydrazone
  • SDA was capable of inhibiting both of the key enzymes of polyamine biosynthesis, thereby eliminating the compensatory response and ensuring complete inhibition of polyamine synthesis.
  • SDA like the other two agents did not inhibit the enzymes of unrelated pathways indicating the specific interaction with polyamines. Similar bioassays may be used by those skilled in the art to identify desired SDA analogues taught herein.
  • Drug T cells Proliferation Leukemic cell growth Ornithine decarboxylase
  • AdoMet decarboxylase Acid phosphatase Alkaline phosphatase Spermine Dialdehyde +++ +++ +++ +++ - - * Difluoro Methyl Ornithine ++ ++ ++++ ⁇ - - ** Bis (ethyl) Spermine +++ +++ ++++ - - - * Metcalf et al ., J. Am. Chem. Soc. 100 , 2551-2553 (1978), ** Porter et al ., Cancer Research 47 , 2821, 1987
  • T and B lymphocytes Human lymphocytes were prepared and separated into T and B lymphocytes as described previously. T and B lymphocytes were incubated with different concentrates of spermine dialdehyde for 1 hour at 37°C. Cells were washed 3 times with RPMI and were then set up in a PHA driven T cell proliferation assay or pokeweed mitogen (pWM) driven B cell proliferation assay. Results are illustrated in Figure 1. As can be seen, spermine dialdehyde inhibited T cell proliferation by 85% at 0.62 x 10 ⁇ 2 mM whereas a much greater quantity was required to suppress B lymphocyte proliferation. Thus, spermine dialdehyde preferentially suppresses T cell proliferation with a much less suppressive effect of B lymphocytes.
  • Bone marrow and spleen cell mixtures (3:1 ratio) from C57BL mice were incubated with control preparation or various concentrations of spermine dialdehyde for 1 hour Cells were then washed extensively and injected intravenously into lethally irradiated histoincompatible AKR mice. Signs of graft vs. host diseases such as hunched back, diarrhea, alopecia, and physical conditions were recorded daily. Survival times of reconstituted mice are shown in Figure 3. Control mice showed severe signs of acute GVHD initiating around 10 days after transplantation. Mice receiving bone marrow treated with spermine dialdehyde manifested only mild signs of GVHD.
  • graft vs. host reaction was generated in the form of popliteal lymph node in F1 recipients by subcutaneous injection of parental spleen cells.
  • Spermine aldehyde was administered intravenously starting 2 days before parental spleen cell injection and continued until the day of lymph node measurement.
  • Increase in popliteal lymph node swelling was scored by difference in weight between test (left) and control (right) lymph node. Results were shown in Figure 4.
  • Substantial swelling was observed in test lymph nodes.
  • Intravenous injection of spermine aldehyde significantly suppressed the GVH reaction as depicted by diminutive popliteal lymph node swelling. This GVH reaction is a typical manifestation of MHC class II reaction involving allogenic T lymphocytes.
  • Spermine dialdehyde when given intravenously suppressed this reaction, suggesting that this molecule mediates the same effect in vivo as in vitro , that is, it acts as a potent suppressor of T cell reactivity.
  • cytotoxic T cells In their spleens which lyse P815 mastocytoma cells bearing DBA specific antigens. Since cytotoxic T cells play an important role in the mediation of organ graft rejection, in vivo suppression of cytotoxic T cell generation may lead to prolonged graft survival.
  • Spermine dialdehyde when administered i.p., suppressed the generation of cytotoxic T cells in a dose related manner ( Figure 5). Dose of 10 mg/kg i.p. appeared to attain maximum suppression which corresponds well with the dose required to achieve maximum suppression of GVH reaction in vivo (5 mg/kg i.v., Figure 4).
  • mice Two pairs of Swiss Webster mice were injected intraperitoneally at 200 mg and 400 mg/kg for toxic effects.
  • Spermine dialdehyde appeared to be acutely irritating in these test animals as indicated by squinting and writhing.
  • Mice receiving 200 mg/kg exhibited no other toxic effect whereas those injected with 400 mg/kg showed abdominal or whole body edema for five days.
  • the 400 mg/kg group showed any positive findings which consisted of a mildly distended abdomen and scab formation at the injection site. This data differed significantly from results reported by Israel et al Supra which showed that the LD100 of this molecule is 40 mg/kg.
  • CsA and all 3 doses of spermine dialdehyde prolonged skin graft survival, especially 10 mg/kg and 20 mg/kg of spermine dialdehyde.
  • mice receiving both i.p. administrations and one injection in the footpad showed significant reduction in swelling when measured 24 hours after challenge and also 48 hours later. Mice receiving 1 injection in the footpad did not exhibit reduction in swelling 24 hours later, but did so 48 hours later.
  • Delayed type hypersensitivity is a T-cell mediated immune response. After immunization with the antigen, mice would be sensitized with specific T cells responding to the antigen. These T cells will migrate to various parts of the body including the footpad. During challenge with the same antigen, these T cells will be reactivated and result in the release of lymphokines, some of which will be chemotactic for monocytes, macrophages and neutrophils. Accumulation of these cells then leads to a local inflammatory response causing substantial swelling. When spermine dialdehyde was administered i.p.
  • Comparative SDA analogues 1-6 listed in the Table below, represent substitution of aldehyde end groups by ester or ketone and reduction in carbon chain length beside the nearest nitrogen. Both methyl and ethyl esters were ineffective in inhibiting the proliferation of T cells/tumor cells or polyamine synthesis.
  • Both methyl ketone (3) and its chloroderivative (4) might show more chemical and metabolical stability than SDA, and one could postulate that they might be suitable for systemic use.
  • preliminary in vitro alkylation studies revealed these compounds to be nonspecific alkylating agents, presumably causing DNA breaks/cross-linking, to result in cell death.
  • the chloride was removed and C-chain was reduced, while retaining the ketone group to yield compounds 5 and 6.
  • the reduction in C-chain from propyl to methyl or ethyl resulted in complete loss of activity (see Table).
  • AdoMetDC methyl-glyoxal-(bisguanylhydrazone)
  • MGBG methyl-glyoxal-(bisguanylhydrazone)
  • a SDA-like compound is ideal for the inhibition of polyamine biosynthesis.
  • in vivo characterization of butyl dialdehyde may also offer further insights into the mechanism of action of SDA.
  • SDA was prepared by acid hydrolysis of the corresponding bioacetal supplied by the Department of pharmaceutical Development at our Institute.
  • [3H]-Thymidine was purchased from New England Nuclear, Boston, MA.
  • Conconavalin A, horse serum, RPMI-1640 and HEPES were from Gibco, Grand Island, NY.
  • NAD and NADP were purchased from Boehringer, Mannheim.
  • Acrolein and disulfiram were obtained from Aldrich Chemical Co., Milwaukee, WI.
  • propionaldehyde and benzaldehyde were from Eastman Kodak Chemical Co., Rochester, NY. Diethyl aminobenzaldehyde (DEAB) was kindly provided by Dr. John Hilton, Johns Hopkins School of Medicine, Baltimore, MD.
  • Murine lymphocytic leukemia cell lines L1210/CPA and L1210/0 (als. known as Le/CPA and Le/0) were kindly provided by Dr. F. Struck, Southern Research Institute, Birmingham, AL. Cells were grown in RPMI-1640, supplemented with 10% horse serum, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin. The cultures were incubated at 37°C in a humidified atmosphere with 5% CO2 in air. Bone marrow cells were flushed from femurs of mice with RPMI-1640, using a tuberculin syringe fitted with a 25 gauge meedle.
  • spleen cells For spleen cells, spleens were removed and passed through a fine wire mesh to get a single homogenous cell suspension. In either case, cells were centrifuged, washed twice and resuspended in the drug exposure medium (see below). Cell viability was determined by trypan blue exclusion and the cell suspension was diluted to the desired concentration.
  • Murine leukemic cells and bone marrow/spleen cells were treated with SDA (1 x 10-6 M to 5 x 10 ⁇ 5 M) in RPMI-1640 or PBS based solution. Wherever mentioned, cells were pretreated with DEAB (10-30 ⁇ M) for 30-60 min at 37°C and then exposed to SDA.
  • [3H]-thymidine incorporation drug or vehicle treated bone marrow or spleen cells were plated in RPMI-1640, containing 10% fetal bovine serum, 10 ⁇ M ⁇ -mercaptoethanol, 25 mM HEPES and 2 mM glutamine. Con-A (4 ⁇ g/ml and 8 ⁇ g/ml) was used as mitogens for bone marrow and spleen cells. Three days later, [3H]-thymidine (0.2 ⁇ Ci/well) was added and cells were harvested after 12-18 hrs. L1210/CPA and L1210/0 cells (0.1 - 1 x 105 cells/well were plated in RPMI-1640 containing 10% horse serum. [3H]-thymidine was added after 24 hrs and cells were filtered on GF/B filter paper for scintillation counting.
  • ALDH activity was measured by monitoring the reduction of NAD or NADp for 5-6 min at 340 nM at 37°C in DU70 spectrophotometer (Beckman).
  • the assay mixture contained 50 mM potassium phosphate buffer, pH 7.4, 2.0 mM NAD (or NADP) and 1.0 mM EDTA.
  • the reaction was started by adding the substrate (SDA or other aldehyde) to the prewarmed (5 min at 37°C) assay mixture containing enzyme preparation. Pyrazole (0.1 mM) was added particularly for liver enzyme, to inhibit alcohol dehydrogenase.
  • Vmax apparent K m and maximum velocity (Vmax) values were calculated using a Lineweaver-Burk plot based on the estimation of the slope by regression analysis.
  • Enzyme activity was expressed as mU (nmoles/min) per mg of protein. Protein contents were determined by the Bradford method, using bovine serum albumin as the standard.
  • Hemopoietic colony forming cells were cultured as described in the art by Iscove & Sieber with minor modifications. SDA/DEAB treated or vehicle treated cells were added to murine hemopoietic culture mixture with erythropoietin and conditioned medium (Terry Fox Laboratories, Vancouver, Canada). The mixture was vortexed and 1-2 ml portions (1 x 105 bone marrow cells) were plated in duplicate in 35 mm plastic petri dishes (lux suspension dishes, Flow Laboratories). The culture dishes were incubated in a humidified atmosphere (95% air : 5% CO2) at 37°C. Colonies were counted on day 8 or 10 for BFU-E, CFU-GM and CFU-mix. Colony types were confirmed with Wright/Giemsa Stain.
  • Cyclophosphamide sensitive L1210/0 cells did not exhibit any detectable enzyme activity with SDA or other aldehydes. Similarly, ALDH activity could not be measured with SDA (up to 2.5 mM) in the mitochondrial fraction of L1210/CPA or mouse liver.
  • Known inhibitors of ALDH were used to confirm the identity of the enzyme involved in the oxidation of SDA (see Table). Disulfiram and DEAB inhibited ALDH activity with mean IC50 values of 4.3 ⁇ M and 0.055 ⁇ M, respectively. Pyrazole, an inhibitor of alcohol dehydrogenase, had no effect on L1210/CPA cytosolic enzyme with SDA as the substrate (data not shown). Acrolein, a highly reactive ⁇ , ⁇ unsaturated aldehyde was also a potent inhibitor of cytosolic ALDH (IC50-2.5 ⁇ M, (see Table).
  • NADP-linked cytosolic ALDH The activity of NADP-linked cytosolic ALDH, measured in L1210/CPA cells with SDA as substrate, was in the range of 3.0-3.5 mU/mg protein.
  • the NADP-linked ALDH was found to be far less sensitive to acrolein than the NAD-linked enzyme (50% inhibition at 50-100 ⁇ M acrolein).
  • DEAB disulfiram and acrolein were very toxic to tumor cells and hemopoietic progenitor cells ex vivo at concentrations much lower than that needed for complete inhibition of ALDH.
  • DEAB besides being the most potent ALDH inhibitor in vivo and in vitro , showed negligible cytotoxicity even up to 50 ⁇ M. Therefore, in subsequent experiments, DEAB was chosen to study the survival of leukemic cells and myeloid progenitor cells after treatment with SDA.
  • L1210/CPA and L1210/0 cells treated with SDA showed a concentration dependent decrease in [3H]-thymidine incorporation.
  • L1210/0 cells appeared to be more sensitive to SDA as compared to L1210/CPA, with mean IC50 values of SDA being 4.66 ⁇ M and 8.3 ⁇ M, respectively.
  • Pretreatment of cells with DEAB (20 ⁇ M) potentiated the cytotoxicitty of SDA in L1210/CPA cells, while having little or no effect on L1210/0 cells.
  • Cell viability estimated immediately after SDA treatment was the same as untreated cells (90%), except for higher concentrations (>25 ⁇ M) of SDA.
  • Murine bone marrow cells treated with SDA in PBS based medium and cultured in RPMI-1640 - 10% FBS for 3 days, showed a dose dependent decrease in [3H]-thymidine incorporation.
  • DEAB (30 ⁇ M) pretreatment to these cells resulted in higher susceptibility at lower concentrations of SDA.
  • ALDH activity could not be detected in homogenates of bone marrow cells. More sensitive conditions, such as specific antibody probes, are suggested to estimate the level of ALDH in these cells.
  • SDA treatment (8-12 ⁇ M) abolished Con A-induced murine spleen cell proliferation, addition of DEAB (20 ⁇ M), however, had no significant effect on the potency of SDA.

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EP89311013A 1988-10-26 1989-10-25 Utilisation des polyamides oxydés, en particulier NN'-Bis-(3-propionaldéhyde)-1-4-diaminobutane (spermine dialdéhyde) comme agents immunosuppresseurs Expired - Lifetime EP0366451B1 (fr)

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AT89311013T ATE103807T1 (de) 1988-10-26 1989-10-25 Verwendung von oxydierten polyamiden, speziell nn'-bis-(3-propionaldehyd)-1-4-diaminobutan als immunsuppressiva.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5374658A (en) * 1988-10-26 1994-12-20 Ortho Phamaceutical Corporation Use of oxidized polyamines, especially NN'-bis-(3-propionaldehyde)-1-4-diaminobutane (spermine dialdehye) in graft treatment
US5386040A (en) * 1992-05-27 1995-01-31 National Starch And Chemical Investment Holding Corporation Low molecular weight polyacetals and polyaldehydes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
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FR2418786A1 (fr) * 1978-03-03 1979-09-28 Nicholas Ind Ltd Acetals et hemiacetals d'aminoaldehyde, et leur utilisation comme medicaments pour inhiber la proliferation des cellules

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5374658A (en) * 1988-10-26 1994-12-20 Ortho Phamaceutical Corporation Use of oxidized polyamines, especially NN'-bis-(3-propionaldehyde)-1-4-diaminobutane (spermine dialdehye) in graft treatment
US5386040A (en) * 1992-05-27 1995-01-31 National Starch And Chemical Investment Holding Corporation Low molecular weight polyacetals and polyaldehydes
US5466862A (en) * 1992-05-27 1995-11-14 National Starch And Chemical Investment Holding Corporation Low molecular weight polyacetals and polyaldehydes
US5612443A (en) * 1992-05-27 1997-03-18 National Starch And Chemical Investment Holding Corporation Low molecular weight polyaldehydes

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AU622720B2 (en) 1992-04-16
JPH02223514A (ja) 1990-09-05
DE68914392D1 (de) 1994-05-11
ES2055094T3 (es) 1994-08-16
EP0366451B1 (fr) 1994-04-06
EP0366451A3 (fr) 1992-01-15
DE68914392T2 (de) 1994-09-15
ATE103807T1 (de) 1994-04-15

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